WO2017016281A1 - Method of detecting target molecule concentration - Google Patents

Abstract

A method of detecting a target molecule concentration comprises the following steps: (1) coupling a first molecular probe to a surface of a magnetic bead to form a magnetic bead probe, and coupling second molecular probes to surfaces of target particles to form target probes; (2) mixing the magnetic bead probe with a target molecule solution so as to capture a target particles at a surface of the magnetic bead probe; (3) adding the target probes, combining the target molecule with the second molecular probes, and forming a magnetic bead probe-target molecule-target probe composite; (4) removing an unreacted target probe; (5) adding an eluent, dissociating a structure of the magnetic bead probe-target molecule-target probe composite into the magnetic bead probe, target molecule, and target probe; (6) extracting a solution obtained from Step (5) to prepare a specimen on a microscope slide, counting and calculating, using a microscope, a total number of target probes, and calculating a target molecule concentration accordingly. The detection method provides increased reliability and sensibility.

Description

Method for detecting target molecule concentration Technical field

The invention relates to a biological detection method, in particular to a method for detecting the concentration of a target molecule.

Background technique

Highly sensitive bioassays are important in the diagnosis and treatment of diseases, especially in the early stages of disease development, in bacterial virus testing, and in clinical basic research. At present, the detection method based on fluorescent label is the main means to achieve higher sensitivity detection. In typical commercial marker detection techniques such as chip arrays, suspension arrays, enzyme-linked immunoamplification (ELISA), target molecules are first immobilized on the surface of the matrix on which the probe molecules are functionalized. Thereafter, a fluorescent molecule or an enzyme having a strong optical signal marks the immobilized target molecule, and the measured optical signal intensity has a positive correlation with the concentration of the target molecule in the sample, thereby realizing the concentration measurement. In the mark detection method, the fluorescent index used therein is generally low in lightness, and the poor light stability makes it extremely easy to cause photobleaching and quenching, and the detection limit is generally not lower than 10 ^-13 mol/L. Although these labeling methods have certain value for many disease detection and diagnosis, they are far from sufficient to meet the early detection needs of some high-mortality diseases. In recent years, high-sensitivity detection technology based on single-molecule detection has received great attention, and it is expected to greatly exceed the traditional detection mode based on fluorescence intensity signal in detection sensitivity. Ordinary single-molecule detection techniques often require expensive instrument support, such as confocal fluorescence microscopy equipped with single photon detection capability, total internal fluorescence microscopy, etc., which are often too expensive in cost and difficult to apply in practical highly sensitive detection. .

Summary of the invention

In order to remedy the above deficiencies of the prior art, the present invention proposes a method for detecting the concentration of a target molecule.

The technical problem of the present invention is solved by the following technical solutions:

A method for detecting a concentration of a target molecule includes the following steps:

(1) coupling a first probe molecule to a surface of a magnetic bead to form a magnetic bead probe, and coupling a second probe molecule to a surface of the labeled particle to form a labeled probe;

(2) mixing, in a reaction vessel, a solution of the magnetic bead probe and a target molecule, the target molecule reacting with the first probe molecule, the target molecule being captured to the magnetic bead probe Forming a target molecule-magnetic bead probe complex on the surface;

(3) adding the labeled probe to the reaction vessel, the target molecule and the second probe molecule Combining to form a magnetic bead probe-target molecule-labeled probe complex;

Or replace the steps (2) and (3) with the following steps (2') and (3'), respectively.

(2') mixing, in a reaction vessel, a solution of the labeled probe and a target molecule, the target molecule reacting with the second probe molecule, the target molecule being captured to the surface of the labeled probe ;

(3') adding the magnetic bead probe to the reaction vessel, the target molecule is combined with the first probe molecule to form a magnetic bead probe-target molecule-labeled probe complex;

(4) removing the labeled probe that is not involved in the reaction;

(5) adding an eluent to the reaction vessel after the step (4), causing the magnetic bead probe-target molecule-labeled probe complex to undergo structural dissociation, dissociation into the magnetic bead probe, The target molecule and the labeled probe;

(6) drawing a solution after the step (5) onto a glass slide to form a sample, counting the labeled probe under a microscope and calculating the total number of labeled probes in the solution, through the total The number of labeled probes is used to calculate the concentration of the target molecule.

Preferably, the number of the magnetic bead probes in the step (2) or the step (3') is 10 ⁵ - 10 ⁷ .

Preferably, the number of the labeled probes in the step (3) or the step (2') is 10 ¹⁰ -10 ¹² .

Preferably, in the step (5), the eluent is added in an amount of 10 to 100 μL.

Preferably, when the detecting method is performed in the order of the step (2) and the step (3), after the end of the reaction of the step (2), the following steps are further included, and then the step is performed ( 3):

(A) introducing a magnet outside the reaction vessel to cause the target molecule-magnetic bead probe complex to collect on one side of the magnet, and sucking the liquid in the reaction vessel without taking the target molecule-magnetic Bead probe complex.

After the step (A), the total volume of the reaction system can be reduced, and then the step (3) can be further carried out, which can further improve the efficiency of the subsequent reaction.

Preferably, the step (4) is specifically: adding a PBS buffer to the reaction vessel after the step (3) or (3') reaction, and then introducing a magnet outside the reaction vessel to make the magnetic beads The probe-target molecule-labeled probe complex is collected on one side of the magnet, and the liquid in the reaction vessel is aspirated to remove the unreacted labeled probe, and the operation is repeated until the unreacted labeled probe is completely Remove.

Preferably, before the sucking of the step (6), the method further comprises the steps of: introducing a magnet outside the reaction vessel after the step (5), and collecting the magnetic bead probe on one side of the magnet.

After the above technical solution, after the magnetic bead probe is first aggregated, the amount of the magnetic bead probe in the solution is reduced, and then the solution is taken up, and then counting under the microscope, the labeled probe is more easily counted, and the working efficiency can be improved.

Preferably, the marking particles are noble metal nanoparticles, fluorescent microspheres or upconverting nanocrystals.

Precious metal nanoparticles (such as gold nanospheres, gold nanorods, gold/silver composite nanoparticles, etc.) have an absorption cross section and a scattering cross section that are 3-5 orders of magnitude higher than ordinary organic fluorescent molecules, and have a very high signal-to-noise ratio. It can be used for highly sensitive detection of many markers. It is especially important that precious metal nanoparticles can easily identify individual particles under ordinary dark field microscopy; similar countable single-particle markers and fluorescent micro-particles Balls (which are formed by coating quantum dots, fluorescent dyes into matrix microspheres) and upconverting nanocrystals, which can also easily achieve single identification under a fluorescence microscope, such single-counting particles due to extremely high letters The noise ratio is much lower than that of a conventional single-molecule detector.

Preferably, the target molecule is a DNA molecule or a protein.

Preferably, the noble metal nanoparticles are gold nanospheres, gold nanorods or gold/silver composite nanoparticles.

The beneficial effects of the invention are as follows: the labeled particles in the invention have a one-to-one correspondence with the target molecules, and the labeled particles can be counted individually under the microscope, and the concentration of the target molecules can be determined by counting the labeled particles, and the single target molecule It also produces an identifiable signal that has a significant sensitivity advantage over the need for multiple markers (corresponding to the target molecule) to produce an identifiable signal in an intensity-based detection method. Furthermore, the liquid phase reaction environment of the present invention The capture kinetics of the target molecule is greatly optimized, so that more target molecules can be captured to the magnetic bead probe, which is beneficial to further improvement of sensitivity. The invention is an ultra-sensitive biological detection method. In one embodiment of the present invention, a genetic sequence of HIV is detected by using a gold nanorod as a labeled probe, and the detection limit can be as low as 1.3×10 ^{− 18} mol/L.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the principle of reaction of a preferred embodiment of the present invention.

2 is an SEM image of a gold nanorod used in a preferred embodiment of the present invention.

3 is an SEM image of a magnetic bead probe-target molecule-labeled probe complex when the target molecule concentration is 100 nM in a preferred embodiment of the invention.

4 is a partial enlarged view of an image of a gold nanorod in a dark field in a preferred embodiment of the present invention.

Figure 5 is a calibration plot of the relationship between target molecule concentration and the average number of gold nanorods in a preferred embodiment of the invention.

detailed description

The invention will now be further described with reference to the drawings in conjunction with the preferred embodiments.

The invention provides a method for detecting a concentration of a target molecule, comprising the following steps:

(1) coupling a first probe molecule to a surface of a magnetic bead to form a magnetic bead probe, and coupling a second probe molecule to a surface of the labeled particle to form a labeled probe;

(2) mixing, in a reaction vessel, a solution of the magnetic bead probe and a target molecule, the target molecule reacting with the first probe molecule, the target molecule being captured to the magnetic bead probe Forming a target molecule-magnetic bead probe complex on the surface;

(3) adding the labeled probe to the reaction vessel, the target molecule is combined with the second probe molecule to form a magnetic bead probe-target molecule-labeled probe complex;

Or replace the steps (2) and (3) with the following steps (2') and (3'), respectively.

(2') mixing, in a reaction vessel, a solution of the labeled probe and a target molecule, the target molecule reacting with the second probe molecule, the target molecule being captured to the surface of the labeled probe ;

(3') adding the magnetic bead probe to the reaction vessel, the target molecule is combined with the first probe molecule to form a magnetic bead probe-target molecule-labeled probe complex;

(4) removing the labeled probe that is not involved in the reaction;

(5) adding an eluent to the reaction vessel after the step (4), causing the magnetic bead probe-target molecule-labeled probe complex to undergo structural dissociation, dissociation into the magnetic bead probe, The target molecule and the labeled probe;

(6) drawing a solution after the step (5) onto a glass slide to form a sample, counting the labeled probe under a microscope and calculating the total number of labeled probes in the solution, through the total The number of labeled probes is used to calculate the concentration of the target molecule.

Among them, the labeled particles refer to particles that can be counted individually under a microscope. The flow chart of the detection principle of the present invention is shown in Fig. 1, wherein: 1 indicates a magnetic bead, 2 indicates a first probe molecule, 3 indicates a target molecule, 4 indicates a labeled particle, and 5 indicates a second probe molecule.

In a preferred embodiment, a known HIV-associated gene (5'-AGAAGATATTTGGAATAACATGACCTGGATGCA-3', a target molecule) is exemplified. The present invention will be described in detail using gold nanorods as labeling particles.

The method for detecting the concentration of a target molecule includes the following steps:

(1) According to the above HIV-related genes, two probe molecules (DNA sequences) are designed according to the conventional method. The first probe molecule is modified with an amino group at the 3' end, and the specific sequence is 5'-TTATTCCAAATATCTTCT-NH2-3. ', coupled to a magnetic bead to form a magnetic bead probe; the second probe molecule is modified with a thiol group at the 5' end, the specific sequence is 5'-HS-TGCATCCAGGTCATG-3', which is coupled to the gold nanorod A labeled probe is formed thereon, and an electron micrograph of the gold nanorod used therein is shown in FIG.

(2) 10 ⁶ beads were added to the reaction probe tube 60 microliters of a solution containing the target molecule (target molecule concentration 10OnM) in one hour, the reaction probe molecules with target molecules of the first, is captured to the magnetic On the surface of the bead probe, after the reaction is completed, a magnet is introduced outside the tube wall, and the liquid in the tube is carefully sucked away without taking away the target molecule-magnetic bead probe complex.

(3) 10 ¹² labeled probes were added to the test tube of step (2), and reacted for 2 hours, and the target molecule was combined with the second probe molecule to form a magnetic bead probe-target molecule-labeled probe complex.

(4) Add 100 μL of phosphate (PBS) buffer to the test tube, shake gently for several times, and then introduce a magnet outside the tube wall to make the magnetic bead probe-target molecule-labeled probe complex on the magnet side. Gather, pipette the liquid from the test tube to remove the unreacted labeled probe, and repeat this step six times to completely remove the incompletely reacted labeled probe. The magnetic bead probe-target molecule-labeled probe complex obtained in this step was made into an SEM sample (as shown in FIG. 3), and it was observed that each magnetic bead had a large number (~7000). The gold nanorods are coated on the surface, which proves that the strong interaction between the double-stranded DNA can indeed form a stable structure between the magnetic beads and the gold nanorods, indicating that the label is reliable, which is the gold nanorod probe available. The experimental basis for label detection.

(5) Add 20 μl of eluent (secondary degranulated water in this example) to the test tube and heat in a water bath at 70 ° C for 5 minutes to compound the magnetic bead probe-target molecule-labeled probe. The structure of the object is dissociated and dissociated into a magnetic bead probe, a target molecule, and a labeled probe.

(6) Introduce a magnet outside the tube wall, carefully pipette 4 μl of the supernatant droplet onto a clean glass slide with a pipette, and cover the microscope with a cover slip to form a microscope sample. The microscope counts the red highlights in the sample, and the dark field micrograph is shown in Figure 4.

The random selection of 30 images can be used to determine the concentration of target molecules. In the experiment, we tested a series of concentrations of target molecule DNA and developed a concentration calibration curve, as shown in Figure 5. According to the definition of the standard deviation of 3 times the detection limit, the lower limit of detection of the method in DNA detection is about 1.3X10 ^-18 mol/L, which is 4-5 orders of magnitude higher than the detection technique based on fluorescence intensity. High application value.

According to the above detection method, the same standard DNA sample of 8×10 ^-17 mol/L was detected and compared with the above-mentioned calibration curve, as shown in Fig. 5, the concentration was 6.7±2.1. ×10 ^-17 mol/L, the error is about 16%, indicating that the detection method of the present invention is highly reliable.

In the above embodiment, the magnetic beads having the superparamagnetic characteristics and coupled with the first probe molecules are used as the capture matrix, and the target molecules in the target molecule solution are captured on the surface of the magnetic beads, which is easy to count single particles. The labeled particles are coupled to the second probe molecule, and the target molecules captured on the surface of the magnetic beads are labeled to form a magnetic bead probe-target molecule-labeled probe complex, which utilizes the superparamagnetism of the magnetic beads. The excess particles participating in the label separate the reaction system, and by elution, the particles participating in the label are released from the surface of the magnetic beads into the solution, and the labeled particles in the solution are counted to determine the concentration of the target molecule. In some other embodiments, the target molecule may be first captured on the labeled particle coupled to the second probe molecule, and then reacted with the magnetic bead coupled to the first probe molecule to form a magnetic bead probe. - Target molecule - labeled probe complex.

In other embodiments, the target molecule can also be a protein, and accordingly, the probe molecule is an antibody and the eluent is a urea solution.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Claims (10)

1. A method for detecting a concentration of a target molecule, comprising the steps of:

   (1) coupling a first probe molecule to a surface of a magnetic bead to form a magnetic bead probe, and coupling a second probe molecule to a surface of the labeled particle to form a labeled probe;

   (2) mixing, in a reaction vessel, the magnetic bead probe and a target molecular solution, the target molecule reacting with the first probe molecule, the target molecule being captured to a surface of the magnetic bead probe Forming a target molecule-magnetic bead probe complex;

   (3) adding the labeled probe to the reaction vessel, the target molecule is combined with the second probe molecule to form a magnetic bead probe-target molecule-labeled probe complex;

   Or replace the steps (2) and (3) with the following steps (2') and (3'), respectively.

   (2') mixing, in a reaction vessel, the labeled probe and a target molecule solution, the target molecule reacting with the second probe molecule, the target molecule being captured to a surface of the labeled probe;

   (3') adding the magnetic bead probe to the reaction vessel, the target molecule is combined with the first probe molecule to form a magnetic bead probe-target molecule-labeled probe complex;

   (4) removing the labeled probe that is not involved in the reaction;

   (5) adding an eluent to the reaction vessel after the step (4), causing the magnetic bead probe-target molecule-labeled probe complex to undergo structural dissociation, dissociation into the magnetic bead probe, The target molecule and the labeled probe;

   (6) drawing a solution after the step (5) onto a glass slide to form a sample, counting the labeled probe under a microscope and calculating the total number of labeled probes in the solution, through the total The number of labeled probes is used to calculate the concentration of the target molecule.
2. The method for detecting the concentration of a target molecule according to claim 1, wherein the number of the magnetic bead probes in the step (2) or the step (3') is 10 ⁵ - 10 ⁷ One.
3. The method for detecting the concentration of a target molecule according to claim 1, wherein the number of the labeled probes in the step (3) or the step (2') is 10 ¹⁰ -10 ¹² .
4. The method for detecting the concentration of a target molecule according to claim 1, wherein in the step (5), the eluent is added in an amount of 10 to 100 μL.
5. The method for detecting a concentration of a target molecule according to claim 1, wherein when said detecting method is carried out in the order of said step (2) and said step (3), the reaction at said step (2) Knot After the bundle, the following steps are also included, and then the step (3) is performed:

   (A) introducing a magnet outside the reaction vessel to cause the target molecule-magnetic bead probe complex to collect on one side of the magnet, and sucking the liquid in the reaction vessel without taking the target molecule-magnetic Bead probe complex.
6. The method for detecting the concentration of a target molecule according to claim 1, wherein the step (4) is specifically: adding a PBS buffer to the reaction vessel after the step (3) or (3') reaction, and then Introducing a magnet outside the reaction vessel to cause the magnetic bead probe-target molecule-labeled probe complex to aggregate on one side of the magnet, and sucking the liquid in the reaction vessel to detect the unreacted label Needle removal, the procedure was repeated until the unreacted labeled probe was completely removed.
7. The method for detecting a concentration of a target molecule according to claim 1, further comprising the step of: introducing a magnet outside the reaction vessel after the step (5), before the sucking of the step (6), The magnetic bead probes are collected on one side of the magnet.
8. The method for detecting a concentration of a target molecule according to any one of claims 1 to 7, wherein the labeled particles are noble metal nanoparticles, fluorescent microspheres or upconverting nanocrystals.
9. The method for detecting a concentration of a target molecule according to any one of claims 1 to 7, wherein the target molecule is a DNA molecule or a protein.
10. The method for detecting the concentration of a target molecule according to claim 8, wherein the noble metal nanoparticles are gold nanospheres, gold nanorods or gold/silver composite nanoparticles.

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